The overall goal of this project is to demonstrate the feasibility of therapeutic cloning in Parkinsonian mice. Stem cell and cloning technologies have received significant public and scientific attention. The promise of stem cells is the generation of unlimited numbers of specific cells for regenerative medicine. Cloning allows the generation of genetically identical organisms. Therapeutic cloning combines these promises towards generating an unlimited source of autologous, specialized cells for transplantation therapy. However, substantial hurdles remain on the path towards that end such as efficient generation of nuclear transfer embryonic stem (ntES) cells, terminal differentiation of ntES cells into specialized cells of therapeutic potential and assessment of in vivo safety and function of ntES cell progeny. Our proposal will address several of these key issues. We have recently succeeded in the generation of ntES cells from adult mice and in coaxing such cells into midbrain dopamine neurons in vitro (Science 2001,292:740-3). Here we propose to derive autoiogous ntES cells from tail tip biopsies of adult Parkinsonian mice. For these studies transgenic Z/EG double reporter mice will be used. These mice constitutively express lacZ but switch to eGFP expression upon Cre-mediated recombination, thus allowing genetic distinction between graft and donor within the context of an autologous graft. Upon derivation MES lines will be subjected to electroporation with the pMC-Cre expression construct. Successful eGFP expressing clones will be selected, proliferated in vitro and converted into dopamine neurons using a previously defined 5-step in vitro protocol. Fluorescence activated cell sorting (FACS) for the neural-specific surface molecule PSA-NCAM will be used to eliminate-any remaining undifferentiated cell populations. Non-invasive dopamine measurements will allow functional screening of ntES progeny prior to implantation. Cells producing adequate levels of dopamine will be transplanted back into the striaturn of the same mice that provided adult cells for ntES generation (autologous grafts). Behavioral and histological parameters will be used to assess the therapeutic function as well as the number and types of surviving ntES progeny in vivo. This study is the first attempt of therapeutic cloning in mice and might pave the way for the application of this approach in other diseases. The proposal is designed in the spirit of the R21 application using innovative strategies to demonstrate the feasibility of novel high-impact techniques in the experimental treatment of Parkinson's disease